Establishing diagnostic cut-off criteria for the COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative test through validation against the Amplicor DNA test v1.5 for infant diagnosis using dried blood spots

Abstract 

Background

As antibody testing cannot confirm HIV-1 infection in children less than 18 months of age, diagnosis in these children depends on nucleic acid testing. The COBAS^® AmpliPrep/COBAS^® TaqMan^® (CAP/CTM, Roche^® Molecular Systems, Inc., Branchburg, NJ) HIV-1 Qualitative test is a total nucleic acid real-time PCR assay utilising whole EDTA blood or dried blood spots (DBS), which recently replaced the Roche^® AMPLICOR^® DNA test v1.5 (Amplicor) as the diagnostic HIV PCR assay in many South African laboratories. For the Amplicor assay, stringent diagnostic criteria were previously formulated for the local population, and a comparison reported the CAP/CTM's sensitivity at 99.7% and specificity at 100% for both sample types compared to these Amplicor criteria.

Objectives

To validate the assay prior to introduction in our laboratory and to define stringent diagnostic cut-off criteria.

Study design

Whole EDTA blood samples from patients younger than 18 months sent for routine HIV-1 diagnosis were tested by Amplicor, and positive results were confirmed from DBS. CAP/CTM assays were subsequently performed from DBS.

Results

The CAP/CTM had a sensitivity of 98.8% and a specificity of 97.1%, but a positive predictive value (PPV) of only 78.7% compared to the Amplicor assay. Samples positive by CAP/CTM but negative by Amplicor displayed poor amplification curves compared to concordant positive samples. Upon re-testing those with sufficient material available by CAP/CTM, all showed negative results.

Conclusions

The decreased PPV may either be due to false positive CAP/CTM results, or increased sensitivity compared to the Amplicor assay. Criteria were formulated for defining presumed false-positive results.

Abbreviations: CAP/CTM, COBAS^® AmpliPrep/COBAS^® TaqMan^®, Ct, cycle threshold, DBS, dried blood spots, EDTA, ethylenediamine tetraacetic acid, HIV-1, Human Immunodeficiency Virus Type 1, IC, internal control, PCR, polymerase chain reaction, SOP, standard operating procedure

Keywords: HIV, PCR, Diagnosis, Dried blood spot, CAP/CTM